Vkorc1 sequencing suggests anticoagulant resistance in rats in New Zealand.

BACKGROUND: Anticoagulant toxins are used globally to control rats. Resistance of Rattus species to these toxins now occurs in at least 18 countries in Europe, America and Asia. Resistance is often associated with single nucleotide polymorphisms (SNPs) in the Vkorc1 gene. This study gives a first overview of the distribution and frequency of Vkorc1 SNPs in rats in New Zealand. New Zealand is unusual in having no native rodents but three species of introduced Rattus - norvegicus Berk., rattus L. and exulans Peale. RESULTS: Sequence variants occurred in at least one species of rat at all 30 of the sites sampled. Three new SNPs were identified, one in kiore and two in ship rats. No SNPs previously associated with resistance were found in Norway rats or kiore, but seven ship rats were heterozygous and one homozygous for the A74T variant. Its resultant Tyr25Phe mutation has previously been associated with resistance to both first- and second-generation anticoagulants in ship rats in Spain. CONCLUSIONS: This is the first evidence of potential resistance to anticoagulant toxins in rats in New Zealand. Further testing using blood clotting response times in dosed rats is needed to confirm resistance potentially conferred by the Tyr25Phe mutation. Assessment is also needed of the potential of the other non-synonymous variants (Ala14Val, Ala26Val) recorded in this study to confer resistance to anticoagulant toxins. © 2016 Society of Chemical Industry.

Diet analysis of small mammal pests: A comparison of molecular and microhistological methods.

Knowledge of what pest species are eating is important to determine their impact on stored food products and to plan management strategies accordingly. In this study, we investigated the food habits of 2 rodents, Rattus rattus (ship rat) and Mus musculus castaneus (house mouse) as well as an insectivore, Suncus murinus (shrew), present in human dwellings. Both a microhistological approach and a DNA barcoding approach were used in the present study. Following DNA extraction, amplification was performed using group-specific primers targeting birds, plants and invertebrates. Resulting polymerase chain reaction products were sequenced and analyzed to identify the different prey species present in the gut contents. The findings from the application of both techniques were in agreement, but the detection of prey type with each technique was different. The DNA barcoding approach gave greater species-level identification when compared to the microhistological method, especially for the invertebrate and avian prey. Overall, with both techniques, 23 prey taxa were identified in the gut contents of the 3 species, including 15 plants, 7 insects and a single bird species. We conclude that with a selection of suitable "barcode genes" and optimization of polymerase chain reaction protocols, DNA barcoding can provide more accurate and faster results. Prey detection from either technique alone can bias the dietary information. Hence, combining prey information of both microhistological analysis and DNA barcoding is recommended to study pest diet, especially if the pest is an omnivore or insectivore species.

Selection on MHC class II supertypes in the New Zealand endemic Hochstetter's frog.

BACKGROUND: The New Zealand native frogs, family Leiopelmatidae, are among the most archaic in the world. Leiopelma hochstetteri (Hochstetter's frog) is a small, semi-aquatic frog with numerous, fragmented populations scattered across New Zealand's North Island. We characterized a major histocompatibility complex (MHC) class II B gene (DAB) in L. hochstetteri from a spleen transcriptome, and then compared its diversity to neutral microsatellite markers to assess the adaptive genetic diversity of five populations ("evolutionarily significant units", ESUs). RESULTS: L. hochstetteri possessed very high MHC diversity, with 74 DAB alleles characterized. Extremely high differentiation was observed at the DAB locus, with only two alleles shared between populations, a pattern that was not reflected in the microsatellites. Clustering analysis on putative peptide binding residues of the DAB alleles indicated four functional supertypes, all of which were represented in 4 of 5 populations, albeit at different frequencies. Otawa was an exception to these observations, with only two DAB alleles present. CONCLUSIONS: This study of MHC diversity highlights extreme population differentiation at this functional locus. Supertype differentiation was high among populations, suggesting spatial and/or temporal variation in selection pressures. Low DAB diversity in Otawa may limit this population's adaptive potential to future pathogenic challenges.

Phylogenetic analysis of New Zealand earthworms (Oligochaeta: Megascolecidae) reveals ancient clades and cryptic taxonomic diversity.

We have constructed the first ever phylogeny for the New Zealand earthworm fauna (Megascolecinae and Acanthodrilinae) including representatives from other major continental regions. Bayesian and maximum likelihood phylogenetic trees were constructed from 427 base pairs from the mitochondrial large subunit (16S) rRNA gene and 661 base pairs from the nuclear large subunit (28S) rRNA gene. Within the Acanthodrilinae we were able to identify a number of well-supported clades that were restricted to continental landmasses. Estimates of nodal support for these major clades were generally high, but relationships among clades were poorly resolved. The phylogenetic analyses revealed several independent lineages in New Zealand, some of which had a comparable phylogenetic depth to monophyletic groups sampled from Madagascar, Africa, North America and Australia. These results are consistent with at least some of these clades having inhabited New Zealand since rifting from Gondwana in the Late Cretaceous. Within the New Zealand Acanthodrilinae, major clades tended to be restricted to specific regions of New Zealand, with the central North Island and Cook Strait representing major biogeographic boundaries. Our field surveys of New Zealand and subsequent identification has also revealed extensive cryptic taxonomic diversity with approximately 48 new species sampled in addition to the 199 species recognized by previous authors. Our results indicate that further survey and taxonomic work is required to establish a foundation for future biogeographic and ecological research on this vitally important component of the New Zealand biota.

Isolation and characterization of microsatellite loci from Lochmaea suturalis, the heather beetle (Coleoptera: Chrysomelidae), a pest in Europe and a biocontrol agent in New Zealand.

The heather beetle Lochmaea suturalis which is native to northwest Europe has been released as a biocontrol agent for heather in New Zealand. We have isolated and optimized eight microsatellite loci from New Zealand beetles. These loci provide markers with high polymorphism ranging from four to 20 alleles per locus. Observed heterozygosity averaged 0.631 per locus. These results suggest the markers are useful for population studies that will contribute to assessment of L. suturalis as a biocontrol agent.

Genome characterization of Botrytis virus F, a flexuous rod-shaped mycovirus resembling plant 'potex-like' viruses.

This study reports the first sequence of a flexuous rod-shaped mycovirus and also the first molecular characterization of a virus that infects the plant-pathogenic fungus BOTRYTIS: cinerea. The mycovirus BOTRYTIS: virus F (BVF) contains an ssRNA genome of 6827 nucleotides and a poly(A) tract at or very near the 3' terminus. Computer analysis of the genomic cDNA sequence of BVF revealed two potential open reading frames (ORFs) encoding proteins of 212 kDa (ORF1) and 32 kDa (ORF2). ORF1 showed significant sequence identity to the RNA-dependent RNA polymerase (RdRp)-containing proteins of plant 'tymo-' and 'potex-like' viruses. However, the ORF1 protein contained an opal putative readthrough codon between the helicase and RdRp regions, a feature not seen in this position in 'tymo-' and 'potex-like' replicases sequenced to date. ORF2 shared amino acid similarity with coat proteins of plant 'potex-like' viruses. Three untranslated regions were present in the genome, comprising a region of 63 nucleotides preceding the initiation codon of ORF1, a 93 nucleotide stretch between ORFs 1 and 2 and a 3'-terminal region of 70 nucleotides preceding the poly(A) tract. The nucleotide sequence of a putative defective RNA (D-RNA) of 829 nucleotides was also determined. The D-RNA contained one potential ORF comprising the N-terminal region of the replicase fused in-frame to the C-terminal region of the coat protein. It is proposed that the mycovirus BVF belongs to a new, as yet unassigned genus in the plant 'potex-like' virus group.